Antibodies have traditionally been used as research tools, and have more recently been developed for use as diagnostics and in drugs and therapeutic applications.
Antibodies are widely utilized for analysis, purification, and enrichment. Research and clinical applications that make use of antibodies are extremely common and encompass a wide variety of subject matters. See Lipman N S, Jackson L R, Trudel L I, Weis-Garcia F. ILAR J. 46(3):258-68. Review (2005). Some applications include immunolocalization, immunoblotting, immunoprecipitation, RIA and ELISA assays, enzyme-linked immunospot assay (“ELISPOT”), proteomics/antibody microarray technology, x-ray crystallography, affinity purification/enrichment, fluorescence-activated cell sorting (“FACS”) analysis, expression library screening, immunofluorescence, immunohistochemistry, immunoimaging, and magnetic-activated cell sorting (“MACS”), although this list is by no means exhaustive, and other applications that make practical use of antibodies are recognized by those skilled in the art.
Both monoclonal and polyclonal antibodies (monoclonal antibodies being preferred) have also proven useful as catalytic agents capable of mediating the catalysis of specific synthetic organic reactions. See Xu Y, Yamamoto N, Janda K D. Bioorg Med Chem 12:5247-5268 (2004).
Antibodies can also be used to modulate cellular activity in the context of cell culture, live animals, or human patients. Antibodies can neutralize/disrupt or activate/stimulate normal cellular signaling by binding their corresponding antigen. In so doing, the antibody may mimic ligand binding and activate a receptor, or block ligand binding by evincing antireceptor activity. Alternatively, the antibody may imitate the mechanism by which many receptors are naturally activated by their ligands, through cross-linking of receptors. For example, incubation of B cells with anti-IgM or T cells with anti-CD3, anti-T cell receptor, or anti-Thy-1 is sufficient to mediate cross-linking of the respective cells surface antigens and stimulate an intracellular signaling cascade, which results in cell growth. See Koike T, Yamagishi H, Hatanaka Y, Fukushima A, Chang J, Xia Y, Fields M, Chandler P, Iwashima M. Biochem Mol Biol 278:15685-15692 (2003). It may be recognized from these examples that given an antibody to a known antigen, antibodies can be used to discern the role of the antigen in cellular function
Antibodies have also demonstrated exceptional promise as drugs and therapeutic agents. After vaccines, antibodies presently constitute the second largest class of drugs and represent the most rapidly growing class of human therapeutics. Carter P J. Nat Rev Immunol. 6(5):343-57 (2006). A widely known example of a therapeutic antibody is infliximab, which is used to neutralize tumor necrosis factor (TNF1)-α in patients, which makes it potentially valuable in treating Crohn's disease. See Kirman I, Whelanand R L, Nielsen O H. Eur J Gastroentero Hepat 16:639-641 (2004).
Antibody-drug conjugates (ADCs) are monoclonal antibodies (mAbs) linked to active molecules, such as drugs, enzymes, or radioisotopes. By employing a rapidly internalizing mAb, one is able to deliver the drug inside target cells. The environment inside the cell cleaves the linker, which releases the drug and allows it to have the desired effect. An example of an antibody-drug conjugate is an antibody linked to a cytotoxic molecule, which kills target cells that possess the components necessary to cleave the molecule-antibody linker. See, e.g., Wu A M & Senter P D. Nat. Biotechnol. 23(9):1137-46 (2005).
Other uses for antibodies in the research, diagnostic, and therapeutic contexts are widely recognized among those skilled in the art, and additional applications continue to be developed. However, while antibodies can target water-soluble regions of antigens, the active domains of many potential targets lie within the hydrophobic biological membrane, into which antibodies cannot penetrate.
Some types of integral membrane proteins include, inter cilia, integrins, cadherins, selectins, NCAM, insulin receptors, and some varieties of cell adhesion and receptor proteins. More generally, membrane proteins may comprise a single transmembrane helix per chain, and in other cases, the membrane protein may comprise a homo- or heterooligomeric protein, each chain thereof having one or more transmembrane helix. The transmembrane region of membrane proteins, which are known to function as any of channels, receptors, enzymes, enablers of cell recognition and/or adhesion, anchors, or energy transducers, commonly possess sections that adopt an alpha-helical configuration. This is because polar CONH groups (peptide bonds) of the polypeptide backbone of transmembrane segments must participate in hydrogen bonds (H-bonds) in order to lower the cost of transferring them into the hydrocarbon interior, and such H-bonding is most easily accomplished with alpha-helices by which all peptide bonds are H-bonded internally. Thus, although the roles of different types of membrane proteins can vary widely, the alpha helix configuration is commonly observed in the transmembrane region.
At present, although methods for designing antibodies for targeting hydrophilic antigens are well developed, there are no corresponding methods for the design of molecules that can be directed against non-water-soluble targets, including the transmembrane region of integral membrane proteins.